Insights into the binding mode of sulphamates and sulphamides to hCA II: crystallographic studies and binding free energy calculations

Figures & data

Figure 1. Structural formulas of topiramate (1), JNJ-26990990 (2), 2-methyl-5-nitro-imidazole-sulphamate (3), 2-methyl-5-nitro-imidazole-sulphamide (4) and the topiramate sulphamide analogue (5). hCA II inhibition constants for compounds 3 and 4 are also reported²⁶.

Table 1. Data collection and refinement statistics. Values in parentheses refer to the highest resolution shell (1.86–1.80 Å).

Crystal parameters
Space group	P21
a (Å)	42.2
b (Å)	41.3
c (Å)	71.7
γ (°)	104.3
Number of independent molecules	1
Data collection statistics
Resolution (Å)	25.3–1.80
Wavelength (Å)	1.54178
Temperature (K)	100
Rmerge (%)^a	3.5 (9.1)
<I>/<σ(I)>	35.8 (10.6)
Total reflections	107,169
Unique reflections	22,183
Redundancy (%)	4.8 (2.7)
Completeness (%)	98.8 (92.9)
Refinement
Resolution (Å)	25.3–1.80
Rwork (%)^b	15.7
Rfree (%)^b	19.5
RMSD from ideal geometry
Bond lengths (Å)	0.012
Bond angles (°)	1.7
Number of protein atoms	2055
Number of water molecules	237
Number of inhibitor atoms	16
Average B factor (Å^2)
All atoms	13.3
Protein atoms	12.1
Inhibitor atoms	16.0
Water molecules	23.2

^a R_merge = Σ_hklΣ_i|I_i(hkl) − <I(hkl)>|/ Σ_hklΣ_iI_i(hkl), where I_i(hkl) is the intensity of an observation and < I(hkl)> is the mean value for its unique reflection; summations are over all reflections.

^b R_work = Σ_hklǁFo(hkl)| − |Fc(hkl)ǁ/Σ_hkl|Fo(hkl)| calculated for the working set of reflections. R_free is calculated as for R_work, but from 5% of the data that was not used for refinement.

Table 2. Partial atomic charges (e) computed for the three ligands in complex_O, complex_N and complex_NO, respectively. Charges were calculated via the RESP fitting procedure as implemented in the PyRED server using Gaussian09 software.

Complex_O		Complex_N		Complex_NO
Ligand atom	Charge	Ligand atom	Charge	Ligand atom	Charge
N1	−1.7264	N1	−1.7369	N1	−1.6903
H1	0.6300	H1	0.5896	H1	0.5976
S1	1.2394	S1	1.4216	S1	1.3758
O1	−0.4723	O1	−0.5319	O1	−0.4851
O2	−0.5586	O2	−0.5903	O2	−0.6095
O3	−0.3736	N2	−0.7886	O3	−0.4657
C1	0.0398	H2	0.4052	C1	0.3176
H11	0.1055	C1	0.2330	H11	0.0483
H12	0.0678	H11	0.0232	H12	−0.0588
H13	0.0484	H12	−0.0722	H13	−0.0300
		H13	0.0473

Figure 2. Active site region in the hCAII/3 complex. Hydrogen bonds, active site Zn²⁺ coordination and residues establishing van der Waals interactions (distance <4.0 Å) with the inhibitor are reported. Sigma-A weighted |2Fo-Fc| simulated annealing omit map (at 1.0 sigma) relative to the inhibitor molecule is also shown.

Figure 3. (A) Structural superposition between hCA II/3 (green) and hCA II/4 (white, PDB code 4MO8)²⁶. (B) Active site region in the hCAII/4 complex. Hydrogen bonds, active site Zn²⁺ coordination and residues establishing van der Waals interactions (distance <4.0 Å) with the inhibitor are reported.

Table 3. Distances between Thr200OG1 atom and the sulphamide N2 atom in hCA II/sulphamide complexes. Only sulphamides of the type R-NH-SO₂NH₂ were considered.

Compound	N2-Thr200OG1 distance (Å)	PDB code
	3.2	4MO8
	3.5	2H15
	3.0	3M2X
	3.2	3MNU
	3.0	4MDG
	3.0	4Q78
	3.4	4MDM
	2.9	4MDL
	5.0	4PQ7
	3.7	5FDC
	3.7	5FDI

Table 4. Distances between Thr200OG1 atom and the sulphamate O3 atom in hCA II/sulphamate complexes.

Compound	O3-Thr200OG1 distance (Å)	PDB code	Compound	O3-Thr200OG1 distance (Å)	PDB code
	4.7	5O07		3.9	2WD2
	4.1	3IBU		4.0	3IBI
	4.2	3IBN		4.1	3IBL
	4.4	1XQ0		4.8	1XPZ
	4.6	3OIM		3.8	3OKU
	4.3	3T85		4.6	3T82
	5.2	2X7T		5.0	2GD8
	4.4	2X7U		4.8	2X7S
	4.6	4ZWY		4.1	4ZX0
	4.7	3DD8		4.5	1TTM
	4.7	3C7P		4.9	3BET
	4.6	3HKU		4.5	1EOU
	4.1	4ZWI		4.8	4R5B
	4.3	3T84		4.7	3T83
	4.5	2WD3

Figure 4. Detail of the active site in the model systems Complex_N (A), Complex_O (B) and Complex_NO (C). In all three cases the ligand, the zinc ion, the three coordinating histidines, Glu106, and enzyme residues giving a major contribution to ligand binding are shown. Only polar hydrogens are shown. Hydrogen bonds are highlighted with red dotted lines, while the distances between O3 and Thr200OG1 are indicated with black arrows.

Table 5. Per-residue binding energy decomposition (given in kcal/mol), calculated by the MM/GBSA method for complex_N, complex_NO and complex_O. Only residues contributing more than −1.0 kcal/mol to the binding are reported.

	ΔGbind-Val143	ΔGbind-Leu198	ΔGbind-Thr199	ΔGbind-Thr200
complex_N	−1.224	−5.536	−1.409	−3.164
complex_NO	−1.177	−5.467	−1.604	−1.290
complex_O	−1.625	−5.209	−1.764	−2.007

Table 6. Individual energy components (kcal/mol) of ΔG_bind-Thr200 calculated by the MM/GBSA method for complex_N, complex_NO and complex_O.

	ΔEvdW^[a]-Thr200	ΔEelec^[b]-Thr200	ΔGGB^[c]-Thr200	ΔGsur^[d]-Thr200
complex_N	−1.430	0.526	−1.249	−1.011
complex_NO	−1.362	2.673	−1.716	−0.885
complex_O	−1.002	1.076	−1.221	−0.860

^[a] van der Waals contribution.

^[b] Electrostatic contribution.

^[c] Generalised-Born solvation contribution.

^[d] Non-polar solvation contribution.

Rami M, Dubois L, Parvathaneni NK, et al. Hypoxia-targeting carbonic anhydrase IX inhibitors by a new series of nitroimidazole-sulfonamides/sulfamides/sulfamates. J Med Chem 2013;56:8512–20.

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